Synthetic oligonucleotides are important diagnostic tools for the detection of genetic and viral diseases. In addition, oligonucleotides and modified oligonucleotides are of interest as therapeutic candidates that inhibit gene expression or protein function. Large scale synthesis of oligonucleotides for use as therapeutic candidates has become increasingly important since FDA approval of an oligonucleotide analog for the treatment of cytomegalovirus (CMV), and several other oligonucleotide analogs are currently in clinical trials. Kilogram quantities of a purified oligonucleotide analog are needed for each clinical trial.
Preparation of an oligonucleotide using phosphoramidite methodology involves condensation of a nucleoside phosphoramidite and a nucleoside or a nascent oligonucleotide. The condensation reaction (also referred to herein as the coupling reaction) requires an activator (alternatively known as a coupling agent) which facilitates the reaction. The most commonly used activator is the nucleophilic activator 1H-tetrazole. However, 1H-tetrazole is explosive and, therefore, can be hazardous to use in large scale syntheses.
1H-tetrazole is a weak acid which protonates the trivalent phosphorus of the phosphoramidite during the first step of activation. A tetrazolide anion then displaces the dialkylamine group (e.g., N,N-diisopropyl amine) of the phosphoramidite during a second slower step to form a tetrazolyl intermediate which then reacts rapidly with the 5′-primary alcohol group of a nucleoside or a nascent oligonucleotide. When sterically hindered phosphoramidites, such as t-butyl-dimethylsilyl protected ribonucleoside phosphoramidites or 2′-O-methylnucleoside phosphoramidites, are used for oligonucleotide synthesis alternative activators are often needed to increase the rate of the coupling reaction. Alternative activators, such as 5-ethylthio-1H-tetrazole, 5-(p-nitrophenyl)-1H-tetrazole, and benzimidazolium triflate, are often more acidic than tetrazole and, thus, accelerate the rate of protonation of the trivalent phosphorous thereby increasing the rate of condensation.
However, since tetrazole, 5-ethylthio-1H-tetrazole, 5-(p-nitrophenyl)-1H-tetrazole, and benzimidazolium triflate are acidic, they can cause premature deprotection of the 5′-hydroxy protecting group of a phosphoramidite monomer which is typically an acid labile group. Premature deprotection can produce oligonucleotide impurities that are one base longer than the desired product (referred to herein as “N+1 impurities”) and are difficult to separate from the desired product. The longer coupling times generally necessary for RNA synthesis and large scale synthesis result in an increase in premature deprotection of phosphoramidites.
Therefore, non-explosive activators that promote condensation of a nucleoside phosphoramidite with a nucleoside or a nascent oligonucleotide and which may be employed without increasing side products are needed in order to make oligonucleotides more readily available for diagnostic and therapeutic use.